Related Experiment Video
Updated: May 24, 2026
08:58
Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Published on: July 5, 2018
Analysis of binding sites on complement factor I using artificial N-linked glycosylation
Jose I Sanchez-Gallego1, Tom W L Groeneveld, Stefanie Krentz
1Department of Laboratory Medicine, Medical Protein Chemistry, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden.
The Journal of Biological Chemistry
|March 7, 2012
Summary
Factor I (FI) regulates complement pathways by degrading C3b and C4b. Mutations in its FIMAC and SP domains significantly impair this function, highlighting their crucial role.
Area of Science:
- Immunology
- Proteomics
- Biochemistry
Background:
- Factor I (FI) is a serine protease essential for inhibiting complement pathways.
- FI degrades activated complement components C3b and C4b, requiring cofactors like Factor H.
- FI comprises a light chain (serine protease domain) and a heavy chain (FIMAC, CD5, LDLr1/2 domains).
Purpose of the Study:
- To elucidate the functional importance of different Factor I domains in complement regulation.
- To identify key protein-protein interaction sites within Factor I using homology modeling and mutagenesis.
- To understand how mutations affect FI's cofactor-dependent degradation of C3b and C4b.
Main Methods:
- Homology-based three-dimensional modeling of Factor I domains.
- Site-directed mutagenesis to create 20 recombinant FI mutants with altered glycosylation sites.
- Biochemical assays measuring FI's degradation of C3b and C4b in fluid and surface phases with various cofactors.
Main Results:
- Mutations in the FIMAC and SP domains severely inhibited FI's ability to degrade C3b and C4b, regardless of the cofactor.
- Alterations in the CD5 and LDLr1/2 domains had a less significant impact on FI's degradation activity.
- The Michaelis constant (K(m)) for small substrates remained unchanged in all mutants.
Conclusions:
- The FIMAC and SP domains are critical for Factor I's enzymatic activity in complement regulation.
- Cofactors likely form similar complexes with FI and C3b/C4b, emphasizing the importance of domain accessibility.
- Accessible FIMAC and SP domains are essential for the proper function of Factor I in inhibiting complement.
Related Concept Videos
01:25Protein Glycosylation
Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
Glycosylation occurs in...
01:24Oligosaccharide Assembly
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...